Central Circuitry in the Jellyfish Aglantha Digitale

The Journal of Experimental Biology 198, 2261–2270 (1995) 2261 Printed in Great Britain © The Company of Biologists Limited 1995 CENTRAL CIRCUITRY IN THE JELLYFISH AGLANTHA DIGITALE I. THE RELAY SYSTEM G. O. MACKIE1 AND R. W. MEECH2 1Biology Department, University of Victoria, Victoria, British Columbia, Canada and 2Department of Physiology, University Walk, Bristol, UK Accepted 9 June 1995 Summary 1. The relay system is an interneuronal pathway in the 3. The relay system can conduct impulses on its own but margin of the jellyfish Aglantha digitale. It excites a second their conduction velocity is greatly increased when interneuronal pathway, the carrier system, and is itself preceded by either pacemaker or ring giant spikes. This excited by pacemaker neurones concerned with slow phenomenon, termed the ‘piggyback effect’, may be due to swimming. It also excites a slow conduction pathway in the extracellular field effects rather than to actions mediated tentacles causing graded, tonic contractions of all the by chemical or electrical synapses. tentacles during slow swimming. 4. Recordings from the epithelial cells that ensheath the 2. The pacemakers, the carrier system and the relay ring giant and outer nerve ring neurones show miniature system all contribute to the production of excitatory synaptic potentials and other events that seem to reflect postsynaptic potentials (EPSPs) in a giant axon that runs events in the nervous system, but no functions can be in the outer nerve ring (ring giant axon). These EPSPs may assigned to them. cause the latter to spike during slow swimming. If it does 5. There is no obvious counterpart to the relay system in so, it will fire tentacle giant axons, producing twitch medusae lacking escape circuitry. contractions of the tentacles. Such contractions probably help to contract the tentacles rapidly at the start of slow Key words: Cnidaria, jellyfish, hydromedusan behaviour, escape swimming. This is an unusual case of a giant axon that swimming, nervous system, nerve ring, giant axon, intracellular normally mediates escape behaviour being appropriated recording, relay system, carrier system, pacemaker, central circuitry, for use during a non-escape activity. Aglantha digitale. Introduction The existence of bundles of neurones in the margins of evidence from electron microscopy, immunocytochemistry, dye hydromedusae has been known since 1850 when Louis Agassiz injection and extra- and intracellular recordings. described them in Bougainvillea superciliaris (see Mackie, We are now in a position to start filling in the picture for 1989a), and the physiological analysis of hydromedusan another jellyfish, Aglantha digitale. Along with other members behaviour, launched by Romanes (1876), has continued to the of the Family Rhopalonematidae (Mills et al. 1985), this present day. It was Passano (1965) who first applied medusa has neuronal pathways specialized for escape electrophysiological techniques to hydromedusae, uncovering a swimming and rapid, twitch-type tentacle contractions in wealth of conduction pathways and pacemaker systems in the addition to systems driving normal ‘slow’ swimming and slow, marginal nerve bundles. These ‘nerve rings’ are now graded tentacle responses. Escape responses are conducted understood to be the animal’s central nervous system in which around the margin by a ‘ring giant’ axon located in the outer all the major pathways interface and interact synaptically as in nerve ring (Roberts and Mackie, 1980). The ring giant elicits the ganglia of higher invertebrates. As the physiological large excitatory postsynaptic potentials in eight ‘motor giant’ analysis proceeded, it became possible to devise increasingly axons located in the subumbrellar myoepithelium (Meech and comprehensive wiring diagrams of the central circuitry in forms Mackie, 1995). EPSPs that exceed threshold give rise to a large such as Stomotoca atra (Mackie and Singla, 1975; Mackie, overshooting sodium-dependent action potential which is 1975) and Polyorchis penicillatus (Spencer, 1978, 1979; rapidly conducted to the swimming muscles, causing a strong Spencer and Arkett, 1984; Arkett and Spencer, 1986). The escape contraction. The ring giant also excites giant axons in Polyorchis work has gone furthest in this direction, synthesizing the tentacles (‘tentacle giants’), causing them to contract rapidly 2262 G. O. MACKIE AND R. W. MEECH during the escape response. The anatomy of the central nervous specialized escape circuitry. By providing a pathway linking system of A. digitale, based on earlier histological studies the neurones responsible for escape and non-escape swimming, (Roberts and Mackie, 1980; Mackie, 1989a,b; Mackie et al. they allow the animal to draw upon an escape component in 1989; Bickell-Page and Mackie, 1991), is depicted in Fig. 1. its normal swimming behaviour. Slow swimming is initiated by rhythmically active pacemaker neurones in the inner nerve ring. These pacemaker neurones also excite the motor giant axons but, unlike the ring Materials and methods giant, their slower, low-amplitude EPSPs (Meech and Mackie, Specimens of Aglantha digitale Müller were caught off the 1995) give rise to a calcium-based action potential that dock at the Friday Harbor Laboratories, Washington, USA, propagates relatively slowly to the swimming muscles and during May, June and July and were kept in the laboratory at produces a weaker muscle response. Thus, the ability of the 7 ˚C. They were dissected in sea water containing about motor giants to conduct two sorts of action potential (Mackie 115 mmol l21 Mg2+, which relaxes the tentacles and prevents and Meech, 1985) is matched by the existence of two sorts of swimming. As even small A. digitale (height 0.9 cm) may have EPSP (Meech and Mackie, 1995). The role of a family of as many as 80 tentacles, each up to 7 mm long, the tentacles potassium channels in regulating the excitability of the axon were cut short close to their bases to prevent them from getting membrane so as to allow separate sodium and calcium spikes in the way of the recording electrodes. Preparations, which has been explored by Meech and Mackie (1993a,b). consisted of a section of margin about 0.6 cm long (one-quarter Each of the two clearly defined forms of swimming has its of the circumference of the animal) and about 0.4 cm in the own specialized neuronal pathway, but the two systems are not radial direction, were pinned out in small Petri dishes lined completely independent and we show in this paper that subtle with Sylgard 184 (Dow-Corning Corp.) using spines of the interactions occur between them. A key mediator of these cactus Opuntia. The most useful spines are about 0.5 mm long. interactions, and the main focus of the present paper, is a ring Glass microelectrodes were used to cut or isolate nerves by of interneurones we call the ‘relay’ system because it relays scoring the neighbouring myoepithelium. Experiments were excitation from the pacemaker neurones to the ring giant axon. conducted in sea water unless otherwise stated. Another interneuronal system involved in exciting the ring Extracellular recordings were made with suction electrodes giant, the ‘carrier’ system, will be discussed in the companion pulled from heated polyethylene tubing. The amplified signals to this paper (Mackie and Meech, 1995). These systems have were filtered and displayed on a storage oscilloscope equipped no known counterparts in hydromedusae that lack the with a wave-form digitizer. They were recorded on Polaroid Fig. 1. (A) Outline drawing of a A medium-sized Aglantha digitale, 1.2 cm bell height. The proportions change somewhat as the animal grows. (B) Perradial B section through the area boxed in A to show marginal nerve centres. The inner and outer nerve rings are bundles of neurones running in the Mesogloea ectoderm at the base of the velum. There are about 800 neurones in a typical cross section, most of them less than Motor giant 1.0 mm in diameter. The motor Tentacle giant, ring giant and tentacle Endoderm giant giant axons are conspicuously larger than other neurones Inner nerve ring running in the same regions. Both the ring giant and the Velum tentacle giants have prominent Tentacle central vacuoles. Nerves are shown which cross the Outer nerve ring Hair mesogloea at the base of the Pad Ring cell velum and connect the outer and cell giant inner nerve rings. The basal processes of the hair cells make contact with the ring giant axon directly. The pad cell sends a long process under the outer nerve ring, contributing to the epithelial ensheathment of groups of neurones. To simplify the picture, the connections passing around the sides of the tentacle that connect the tentacle giant axon to the outer nerve ring have been omitted along with the small nerve bundle that runs beside the motor giant and the small-diameter nerve plexuses running in the outer velar ectoderm and tentacle ectoderm. The relay system in a jellyfish 2263 film. Most events recorded extracellularly were captured A within the 1 Hz to 1 kHz waveband. Signals were also recorded on an instrumentation tape recorder and/or a chart recorder. Unless otherwise stated, negative potentials are in the upward direction in the extracellular records. It should be noted that an Motor giant electrode on one side of the margin can pick up events from axon both nerve rings. For intracellular recordings, glass capillary micropipettes, RU 21 filled with 3 mol l potassium chloride, were mounted on Inner nerve ring Zeiss (Jena) manipulators fitted with magnetic bases. Amplification of the intracellular signal was conventional and Velum provided for intracellular current injection via a bridge circuit. Stimuli were also applied externally through small co-axial Stimulus bipolar metal electrodes (Clark Electromedical Instruments, RL SNE-100). The preparation was viewed with a stereoscopic binocular BC microscope mounted over a special transillumination base incorporating a double mirror system (Mackie, 1976a). Preparations were kept cool (10–12 ˚C) during recordings using a Cambion thermoelectric cooling unit with a doughnut- shaped cooling platform that allowed transillumination of the 20 mV specimen during recordings.

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